Investigation Of Intramolecular Synergy Of Modular Cellulase GH9

Cellulase plays an important role in the conversion of lignocellulose.Most cellulases are typical modular enzymes,usually composed of one or two catalytic domains(CD)and one or several carbohydrate-binding modules(CBMs),which are linked to the catalytic domains by highly flexible linker(linker).CBM can target the catalytic center of the enzyme adsorbed on the surface to the insoluble cellulose substrate,increase the effective enzyme concentration on the surface of the substrate,and then transport the substrate to the catalytic crack of the enzyme for degradation,thereby improving the enzyme activity.The linker separates CD and CBM by an appropriate distance,which facilitates the dynamic adsorption of CBM on cellulose substrate and increases the catalytic rate.However,the intramolecular synergistic mechanism of modular cellulases remains unclear.Two GH9 endoglucanases,Ac Cel9A and Ac Cel9B,derived from Acidothermus cellulolyticus 11B,are typical modular cellulases.Ac Cel9A is composed of three domains,the N-terminal to C-terminal are the catalytic domain(GH9-CD),CBM3,CBM2;Ac Cel9B is composed of five domains,except for the signal peptide region,which are the catalytic domain(GH9-CD),CBM3,CBM3,FN3,CBM2.In order to clarify the synergistic mechanism between Ac Cel9A and Ac Cel9B modules,the following studies were carried out in this paper:(1)A series of primers were designed using the A.cellulolyticus 11B genome as a template,and mutants containing different modules were obtained by PCR,which were constructed in the vector p ET-28a(+)and heterologously expressed in E.coli.The results showed that,except for the recombinant protein Ac Cel9B-CD-L1,other recombinant proteins could be successfully purified by nickel column affinity chromatography.(2)The characterization of the enzymatic properties of the recombinant proteins showed that the optimum p H of all enzymes was4.0-5.5,indicating that their auxiliary domains had little effect on the optimum p H of the enzymes;The optimum temperature of wild-type and recombinant enzymes containing auxiliary could reach 75°C,while the optimum temperature of Ac Cel9A-CD-L1 with a single catalytic domain was only 60°C,indicating that these auxiliary modules contributed to the thermal stability of the enzyme;The analysis found that CBM3c which are adjacent to the catalytic domain and FN3,could improve the thermal stability of the enzyme,while the CBM2 module was not conducive to the thermal stability of the enzyme;CBM3 played a key role in the efficient degradation of insoluble substrates by modular enzymes,auxiliary modules CBM2 and FN3 could affect the substrate specificity of the enzymes;SEM observation and analysis showed that when CBM2 at the C-terminus of Ac Cel9B was deleted,the modular cellulase Ac Cel9B-(CBM3)₂+FN3 were more likely to destroy the amorphous area of filter paper and increase the degree of loosening between fiber bundles.(3)In order to analyze the molecular mechanism of the synergy between the auxiliary module and the catalytic domain,a series of mutations were made to the linker1 and linker2 of the modular enzyme Ac Cel9A,and the heterologous expression was carried out in E.coli.linker1,which was in close proximity to the catalytic domain,played an important role in protein expression and folding.In order to elucidate the molecular mechanism of the intramolecular synergy of modular cellulases,in this paper,two modular GH9 cellulases from the acidophilic heat-tolerant bacterium A.cellulolyticus 11B were studied,and a series of mutants containing different auxiliary modules were constructed.It was found that each accessory domain played different roles in recombinant protein expression,thermostability,and substrate selectivity.In the process of dissecting the molecular mechanism of cooperation between modules,it was initially found that the linker between the catalytic domain and CBM3c was very important for the soluble expression and correct folding of recombinant proteins.The above research results laid a foundation for the in-depth analysis of the molecular mechanism of the intramolecular synergy of the modular cellulase GH9,and also provided a theoretical basis for the rational design of the modular cellulase.